Papillomaviruses (PVs) infect the epithelia of animals and man, where they generally induce benign proliferation at the site of infection. However, there is a strong association between malignant progression of human genital, anal and oropharyngeal lesions and certain human papillomavirus (HPV) types, most frequently HPV 16. Our research is primarily concerned with development of vaccines and other agent to prevent and treat HPV infections and the neoplasia they induce, the elucidation of the HPV life cycle, and using the insights obtained in these studies to develop treatments against other cancers and chronic diseases. We have developed a simple and efficient strategy for generating papillomavirus-like particles (VLPs) and high titers of infectious papillomavirus particles that transduce encapsidated marker plasmids, i.e. pseudovirions. We have exploited these technologies in our basic virologic and translational research efforts. We have used our pseudovirus technology to develop the first cervicovaginal challenge model for HPVs and used this assay to define the molecular mechanism used by HPV to infect its target tissue and to determine how the antibodies induced L1- and L2-based prophylactic vaccines prevent infection. Key to the process is an obligatory binding to the heparan sulfate proteoglycans (HSPGs) on the basement membrane of a disrupted epithelium. Our development of a method to induce efficient HPV pseudovirus infection of the female genital tract after transient disruption with the over-the-counter spermicide nonoxonol-9 has proven to be the key to our recent development of an effective, and we believe practical, intravaginal vaccination strategy. We have found that intravaginal pseudovirus vaccination of N-9 treated mice induces strong systemic and mucosal T and B cell responses to target antigens transduced by the pseudovirions. Most of the induced T are long lived intraepithelial tissue resident effector memory CD8 T cells (Trms). Critically, CD8 IEL's were not induced after systemic vaccination with peptides or viral vectors. Intravaginal pseudovirus vaccination is a promising approach for focusing immune responses to the female genital tract and so should increase the effectiveness of vaccines directed against HSV infections and against HPV induced neoplasia. In collaboration with Dr. Jeff Cohen, NIAID, vectors expressing HSV antigens are were tested in mouse and guinea pig HSV-2 challenge models. Intravaginal vaccination with these vectors reduced local pathology induced by intravaginal HSV inoculation, but standard parenteral vaccination did not. In a CRADA collaborated with Crucell/Janssen, we have determined that Ad26 and Ad35 vectors induce similar locals T cell responses as intravaginal delivery of HPV pseudovirions and are superior at inducing systemic T cell responses. Crucell has extensive expertise in GMP production of their adenovirus vectors, and the company is moving our findings toward a clinical trial. To more generally evaluate the potential of HPV pseudoviruses as gene transfer vehicles, we conducted a broad infection tropism survey. In patent pending studies, we demonstrated that intact murine epithelium at all sites, whether simple, columnar, or squamous, was highly resistant to both virion binding and infection, whereas disrupted epithelium was susceptible. In contrast, virtually all human-derived carcinoma and melanoma cell lines in the NC1-60 panel were highly susceptible to VLP/psuedovirus binding/infection in vitro. The remarkable specificity of HPV VLP/pseudovirus binding and infection is mediated by specific HSPG modifications on the tumor cell surfaces that mimic those normally found on the basement membrane. The results suggest that HPV VLPs/pseudovirions may be useful in tumor diagnostic or tumor-directed cytotoxic gene or drug conjugate applications. In proof of concept studies, we documented highly specific binding and infection, and dramatic imaging, of human ovarian tumor nodules implanted in nude mouse peritoneum after intraperitoneal injection of RFP-expressing pseudovirus. In a study using a mouse model of ovarian metastases, intraperitoneal injection of Herpes TK-expressing HPV psuedovirions followed by ganciclovir treatment, increased survival of tumor bearing mice. A CRADA with Aura Biosciences was initiated to facilitate further development and clinical testing of this approach to tumor therapy. Based on earlier antibody-based studies by CCR's Peter Choyke, we are investigating tumor imaging and killing by capsids coupled to an infrared dye, IR700. Tumor therapy studies of the dye-coupled VLPs in several mouse models and particularly in a rabbit zenograft model of human uveal melanoma have produced exceptionally encouraging results. These studies have led to an FDA approved phase 1 trial for treatment of ocular melanoma. The treatment phase of the dose and frequency escalation trial has been completed, and we are now collecting one year follow-up safety and efficacy data. In other preclinical studies, for which we a obtained an NCI Director's Innovation Award, we are testing the hypothesis that preexisting immune responses to CMV can be effectively direct to tumors by expressing viral antigens specifically in the tumors via pseudovirus transduction. In order to further investigate the molecular mechanisms of HPV virion assembly and develop a psuedovirus production system that is amenable to industrial scale GMP production, we devised and refined methods for generating DNA plasmid transducing pseudovirions in a cell-free system. The initial basic mechanistic findings were first published. More recently, we have published methods to generate titers of pseudovirions in chemically defined cell-free reactions that rival the titers generated in our optimized intracellular assembly reactions. In unpublished studies, we have discovered methods to efficiently encapsiding RNA molecules. These advances should facilitate the translation of our preclinical vaccine and tumor treatment studies into additional human trials. A long-standing collaboration with DCEG colleagues has resulted in many publications related to the NCI-sponsored prophylactic HPV vaccine clinical trial in Costa Rica (CVT), in the past year focusing on further refinements of the efficacy and safety analyses. Based on recently published post hoc analyses from CVT indicating that even a single dose of Cervarix induces long term protection, we have worked over the last year with our DCEG colleagues to design, secure NCI and Gates funding, and initiate an efficacy trial of one versus two doses of Cervarix and Gardasil-9 in Costa Rican girls. In collaboration with colleagues at McGill University, we have published the results of the first randomized control clinical study demonstrating that topical application of a carrageenan gel protects young women from genital HPV infection.